Cochlear transduction via cerebrospinal fluid delivery of AAV in non-human primates
Authors: Paul T. Ranum, Luis Tecedor, Megan S. Keiser, Yong Hong Chen, David E. Leib, Xueyuan Liu, Beverly L. Davidson
Journal: Molecular Therapy (2023)

Adeno-associated viruses (AAVs) have been approved for clinical use for several indications, 1 Kuzmin D.A. Shutova M.V. Johnston N.R. Smith O.P. Fedorin V.V. Kukushkin Y.S. van der Loo J.C.M. Johnstone E.C. The clinical landscape for AAV gene therapies. Nat. Rev. Drug Discov. 2021; 20: 173-174https://doi.org/10.1038/d41573-021-00017-7 Crossref PubMed Scopus (193) Google Scholar ,2 Bennett J. Maguire A.M. Lessons learned from the development of the first FDA-approved gene therapy drug, voretigene neparvovec-rzyl. Cold Spring Harb. Perspect. Med. 2022; : a041307https://doi.org/10.1101/cshperspect.a041307 Crossref PubMed Scopus (2) Google Scholar with continued progress to evolve new forms for improved targeting of the organ or cell of interest. 3 Goertsen D. Goeden N. Flytzanis N.C. Gradinaru V. Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid. Mol. Ther. Methods Clin. Dev. 2022; 26: 331-342https://doi.org/10.1016/j.omtm.2022.07.010 Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar ,4 Gonzalez T.J. Simon K.E. Blondel L.O. Fanous M.M. Roger A.L. Maysonet M.S. Devlin G.W. Smith T.J. Oh D.K. Havlik L.P. et al. Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing. Nat. Commun. 2022; 13: 5947https://doi.org/10.1038/s41467-022-33745-4 Crossref PubMed Scopus (9) Google Scholar ,5 Davidsson M. Wang G. Aldrin-Kirk P. Cardoso T. Nolbrant S. Hartnor M. Mudannayake J. Parmar M. Björklund T. A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism. Proc. Natl. Acad. Sci. USA. 2019; 116: 27053-27062https://doi.org/10.1073/pnas.1910061116 Crossref PubMed Scopus (90) Google Scholar As our laboratory is interested in CNS gene therapies, 6 Keiser M.S. Ranum P.T. Yrigollen C.M. Carrell E.M. Smith G.R. Muehlmatt A.L. Chen Y.H. Stein J.M. Wolf R.L. Radaelli E. et al. Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain. Nat. Med. 2021; 27: 1982-1989https://doi.org/10.1038/s41591-021-01522-3 Crossref PubMed Scopus (18) Google Scholar we perform routine assessments of transduction distribution of wild-type and evolved AAVs following delivery to the brain. Recently, we included the evaluation of the inner ear in our analyses. To our surprise, we observed striking cochlear inner hair cell transduction (Figure 1, column 1) after intracerebroventricular (ICV) injection of AAV9.EGFP to a Macaca mulatta (rhesus macaque). We find that auditory inner hair cells, cells of the spiral ligament, and cells of the spiral limbus were transduced, with the most robust transduction in the apex of the cochlea (Figure 1, column 1). There were few inner hair cells transduced in the middle and basal turns (Figure 1, column 1). Upon dissection and evaluation of cochlear tissues after similar studies evaluating an AAV9 capsid variant (AAV9.KGG.mNG) after CSF delivery to a Chorocebus aethiops (African green) monkey, there was notable transduction at the apical and upper-middle turns of the cochlea, but the most extensive transduction was observed in the lower-middle and basal turns (Figure 1, column 2). Further, AAV1 and AAV2 capsid variants expressing different fluorophores, AAV1.RPG.mNG and AAV2.HDG.mTFP, transduced nearly all inner hair cells via this route of administration (Figure 2, top columns 1 and 2). AAV9.mCherry, which was pooled with the AAV1 and AAV2 variants under study, performed less well in African green than was observed for AAV9.EGFP in the rhesus macaque, a difference that is potentially indicative of physiological differences between the two non-human primate (NHP) species or experimental variation. (Figure 2, top column 3). Because the transduction patterns of AAV1.RPG.mNG and AAV2.HDG.mTFP were similar, we confirmed expression of the mNeonGreen and mTFP RNA by fluorescence in situ hybridization (FISH). Confocal imaging of FISH probes confirmed highly similar tropism of the AAV1.RPG.mNG and AAV2.HDG.mTFP capsids (Figure 2, bottom columns 1–4). Figure 2AAV1.RPG.mNG and AAV2.HDG.mTFP transduce African green cochlea via CSF Show full caption A single ICV infusion of pooled capsids AAV2.HDG.mTFP, AAV1.RPG.mNG, and AAV9.mRuby3 to a male African green at a dose 3E13 total vector genomes per capsid. The animal was between 4 and 6 years and weighed 6.90 ± 0.68 kg. Columns 1–4: direct fluorescence from AAV2.HDG.mTFP, AAV1.RPG.mNG, and AAV9.mRuby3 in the rhesus macaque cochlea. Row 1: full apical turn. Rows 2–5: transduction at the indicated tonotopic position. Rows 6–9: FISH analysis for mTFP, mNG, and mRuby3 mRNAs. AAVs derived from AAV1 (AAV1.RPG.mNG) had a peptide insert of RPGREAS at aa position 590 in loop 8. The AAV2-derived capsid (AAV2.HDG.mTFP) had a peptide insert of HDGGASR at aa position 587 in loop 8. Wild-type AAV9 (AAV9.mRuby3) was also used. The animal was euthanized 1 month after AAV capsid infusion. Scale bar for direct fluorescence, whole apical turn images is 200 μm. Scale bar for the direct fluorescence apex, upper middle, lower middle, and base images is 50 μm. The scale bar for the FISH apex, upper middle, lower middle, and base images is 50 μm. View Large Image Figure Viewer Download Hi-res image A single ICV infusion of pooled capsids AAV2.HDG.mTFP, AAV1.RPG.mNG, and AAV9.mRuby3 to a male African green at a dose 3E13 total vector genomes per capsid. The animal was between 4 and 6 years and weighed 6.90 ± 0.68 kg. Columns 1–4: direct fluorescence from AAV2.HDG.mTFP, AAV1.RPG.mNG, and AAV9.mRuby3 in the rhesus macaque cochlea. Row 1: full apical turn. Rows 2–5: transduction at the indicated tonotopic position. Rows 6–9: FISH analysis for mTFP, mNG, and mRuby3 mRNAs. AAVs derived from AAV1 (AAV1.RPG.mNG) had a peptide insert of RPGREAS at aa position 590 in loop 8. The AAV2-derived capsid (AAV2.HDG.mTFP) had a peptide insert of HDGGASR at aa position 587 in loop 8. Wild-type AAV9 (AAV9.mRuby3) was also used. The animal was euthanized 1 month after AAV capsid infusion. Scale bar for direct fluorescence, whole apical turn images is 200 μm. Scale bar for the direct fluorescence apex, upper middle, lower middle, and base images is 50 μm. The scale bar for the FISH apex, upper middle, lower middle, and base images is 50 μm.